In addition to their role in countercurrent exchange, descending vasa recta (DVR) are resistance vessels through which blood flow to the renal medulla is supplied. The general hypothesis of this program project grant is that a fundamental mechanism that leads to hypertension is enhanced production of reactive oxygen species (ROS). The general hypothesis to be tested in this subproject is that, during hypertension, increased ROS production in the medulla leads to reduction of nitric oxide (NO) bioavailability and enhanced vasomotor tone of DVR. Preliminary data shows that angiotensin II (AngII) enhances ROS formation in DVR and nephrons and that blockade of ROS formation increase DVR NO formation and blunts vasoconstriction. We will explore our central hypothesis our central hypothesis with the following specific aims. In aim 1 we will test the hypothesis that ROS formation modulates NO generation in outer medullary DVR (OMDVR) isolated from the vascular bundles and outer medullary nephrons isolated from the interbundle region. As an index of NO bioavailability, we will test the effect of AngII induced hypertension on OMDVR vasodilation by acetylcholine. We will also directly measure ROS and NO generation ROS and NO generation by OMDVR and interbundle nephrons using fluorescent probes. In aim 2 we will determine which of the enzymes responsible for ROS and NO generation is modulated during AngII induced hypertension. We will measure NADPH oxidase, eNOS, nNOS and ecSOD expression in OMDVR and nephrons by real time PCR, western blot of outer medullary homogenates, in situ hybridization and immunocytochemistry. We will also test the effect of NADPH oxidase, eNOS and nNOS gene deletion in ROS and NO generation. In aim3, we will test the hypothesis that hypertension is accompanied by a decreased in oxidant defense mechanisms. In aim 3, we will test the hypothesis that hypertension is accompanied by a decreased in oxidant defense mechanisms. We will examine the effects of gene deletion of extracellular superoxide dismutase and the dopamine D5 receptor on ROS generation and NO bioavailability. We will also determine the extent to which the D5 receptor acts through hemeoxygenase.